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Journal of Virology, November 1998, p. 9034-9044, Vol. 72, No. 11
Department of Microbiology and Immunology,
The Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033,1 and
ABL-Basic
Research Program, NCI-Frederick Cancer Research and Development
Center, Frederick, Maryland 217022
Received 11 May 1998/Accepted 4 August 1998
The Gag proteins of Rous sarcoma virus (RSV) and human
immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger
motifs and function to provide the proper density to viral particles.
There are two zinc fingers and at least two I domains within these Gag
proteins. To more thoroughly characterize the important sequence
features and properties of I domains, we analyzed Gag proteins that
contain one or no zinc finger motifs. Chimeric proteins containing the
amino-terminal half of RSV Gag and various portions of the carboxy
terminus of murine leukemia virus (MLV) (containing one zinc finger)
Gag had only one I domain, whereas similar chimeras with human foamy
virus (HFV) (containing no zinc fingers) Gag had at least two.
Mutational analysis of the MLV NC sequence and inspection of I domain
sequences within the zinc-fingerless C terminus of HFV Gag suggested
that clusters of basic residues, but not the zinc finger motif residues
themselves, are required for the formation of particles of proper
density. In support of this, a simple string of strongly basic residues
was found to be able to substitute for the RSV I domains. We also
explored the possibility that differences in I domains (e.g., their
number) account for differences in the ability of Gag proteins to be
rescued into particles when they are unable to bind to membranes.
Previously published experiments have shown that such membrane-binding
mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with
RSV-MLV chimeras now map this difference to the NC sequence of MLV.
Importantly, the same RSV-MLV chimeras could be rescued by
complementation when the block to budding was after, rather than
before, transport to the membrane. These results suggest that MLV Gag
molecules begin to interact at a much later time after synthesis than
those of RSV and HIV.
Retroviral Gag proteins are capable
of directing the production of virus-like particles that are similar in
size, shape, and buoyant density to authentic virions. Three small
regions of the Gag polyprotein, termed assembly domains, have been
found to be important for this budding process (see Fig. 1A)
(35). The membrane-binding (M) domain, located within the
matrix (MA) sequence, is required for the targeting and binding of the
Gag protein to the cytoplasmic face of the plasma membrane
(29). The interaction (I) domain, located within the
nucleocapsid (NC) sequence, provides a major region of interaction
between the 1,500 to 2,000 Gag proteins that make up an individual
particle (30) and mediates the production of particles of
the proper density (1, 33). The late (L) domain, located in
different positions in different Gag proteins, is responsible for a
budding event which occurs after the Gag molecules have reached the
membrane but before the nascent particle is released from the cell
(i.e., during the virus-cell separation step) (20, 34). The
primary focus of this report is the mechanism of I domain function.
In addition to I domains, the NC region of most retroviral Gag proteins
contains one or two zinc finger motifs, characterized by the
amino acid sequence CX2CX4HX4C,
which are known to be involved in viral RNA packaging (3).
Unlike zinc fingers, I domains cannot be identified by a particular
sequence motif, but their presence can be confirmed by analyzing the
density of particles produced upon expression of a given gag
allele. Isopycnic sucrose gradient centrifugation has been used to map
the positions of at least two I domains in the NC regions of human
immunodeficiency virus (HIV) and Rous sarcoma virus (RSV) (1,
33). Interestingly, both of these Gag proteins have the same
number of zinc finger motifs as they do identifiable I domains. Equally
interesting is the conspicuous absence of zinc finger motifs in the
putative NC region of spumavirus Gag proteins (11). To date,
no spumavirus NC region has been analyzed for the presence of specific
sequences capable of controlling the density of virus-like particles.
Although there has been a correlation between the number of zinc finger
motifs in the NC portion of Gag and the number of identifiable I
domains, previous experiments demonstrating that substitutions in the
single zinc finger of murine leukemia virus (MLV) do not alter
particle density (13) led us to believe that there is no
functional significance to this relationship. The experiments described
here confirm this hypothesis, identify dual I domains in the human
foamy virus (HFV) Gag protein, and implicate basic residues as the
primary functional components of I domains. They also suggest that I
domains may play a role in the timing and location of the initial Gag
protein multimerization.
Previously constructed gag alleles.
The RSV
gag gene was obtained from pATV-8, an infectious molecular
clone of the RSV Prague C genome (28). The MLV
gag and pol genes were from pRR88, a clone of the
integrated Moloney MLV proviral genome in the plasmid pGCcos3neo
(13). The HFV gag gene was obtained from pHSRV13,
an infectious molecular clone constructed and kindly
provided by R. Flugel (18). Several of the plasmids used in
these experiments have been previously described, namely, pSV.Myr1
(referred to here as pSV.R.M1) (36), pSV.T10C.PR Newly constructed gag alleles.
Two plasmids were
constructed to express the MLV Gag protein. The vector pSV.MLV, which
expresses the gag and pol genes of MLV, was
constructed by transferring the complete MLV coding sequence (including
env) from pRR88 into pSV.R.M1 in place of the RSV
gag sequence. The MLV fragment was prepared by digesting
pRR88 with AatII (nucleotide [nt] 362), blunt-ending the
resulting 3' overhang with the Klenow fragment of DNA polymerase I
(Klenow), and then digesting it with BssHII (nt 8211). This
was then ligated with T4 DNA ligase to the large fragment of pSV.R.M1,
which had been digested with SstI (nt 255), treated with
Klenow, and digested with BssHII (nt 2724). A plasmid
expressing MLV Gag and a truncated form of Gag-Pol, pSV.MLV.PR
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Importance of Basic Residues in the Nucleocapsid
Sequence for Retrovirus Gag Assembly and Complementation
Rescue
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(34), pSV.Bg-Bs (32), pSV.p25 (33),
and pSV.p25.AD3 (33). Standard protocols were used for
all DNA manipulations (25), and all plasmids were propagated
in Escherichia coli DH-1 cells in 2× YT medium containing
25 µg of ampicillin/ml.
, was
made by first digesting the parent plasmid, pSV.MLV, with
DraIII (MLV nt 2330, 5602, and 6656) and treating with
Klenow. The 10,078-nt fragment was gel purified and recircularized at a
concentration of 20 µg/ml in the presence of XbaI linkers
(5'-CTAGTCTAGACTAG-3'),
which contain termination codons (underlined) in all three
reading frames.
was digested with
PstI (MLV nt 738), treated with Klenow, and digested with
EagI (pSV.R.M1 nt 3658). The 4,901-nt fragment was then
gel-purified and ligated to the 5,774-nt fragment of pSV.R.M1 that had
been prepared with MluI, Klenow, and EagI in
succession, to create pSV.M.M1.PR. pSV.M.M1 was constructed to
express the MLV Gag and Gag-Pol proteins containing the Src
membrane-binding domain in place of the M domain of MLV. For this, the
2,227-nt XhoI-BssHII fragment of pSV.M.M1.PR was replaced with the 6,553-nt XhoI (nt
1559)-BssHII (nt 8211) fragment from pSV.MLV. An
unmyristylated form of the Src-RSV Gag chimera, R.M(), was
constructed by mutagenesis of the R.M1 gag allele by using
previously described methods and an oligonucleotide with the sequence
5'-GGATCAAGCATGGAATCCAGCAAAAGC-3'. As a result, the second codon was changed from GGA (glycine) to GAA (glutamic acid)
and the expression plasmid was named pSV.Myr1(). pSV.MLV.Myr(), which expresses a myristate-minus form of the Src-MLV chimera, was then
constructed by joining the 7,473-nt fragment from pSV.MLV that had been
prepared with PstI, Klenow, and BssHII with the 6,708-nt fragment derived from pSV.Myr() that had been digested with
MluI, Klenow treated, and digested with BssHII.
), or an internally deleted chimera, pSV.T10M.PR, were
created in a similar fashion, except that the parental plasmids
used were pSV.Myr() and pSV.BgM, or pSV.T10C.PR and
pSV.MLV.PR, respectively. The MLV protease (PR) active site
mutation D32L found in pRR88.2204 (12) was subcloned into
pSV.BgM by digesting each plasmid with XhoI and treating it
with Klenow and BssHII. The desired fragments were gel
purified and ligated to form pSV.BgM.PR. To create the point
mutations (pSV.BgM.C26/29S and pSV.BgM.Y28S), internal deletions
(pSV.BgM.
K8-R11 and pSV.BgM.R16-R23), and the
carboxy-terminal truncations (pSV.BgM.S402**, pSV.BgM.A478**,
pSV.BgM.D501**, and pSV.BgM.R521**) of MLV NC, the respective pRR88
mutants were subcloned into pSV.BgM by the same procedure as was used
for the protease active-site mutation (see reference
13 for C26/29S and Y28S and reference
22 for K8-R11 and R16-R23; R521** has been
previously referred to as R44Ter [22] and S402**,
A478**, and D501** are previously unpublished).
To investigate the sequence requirements for dense particle formation,
two constructs which expressed proteins encoding foreign amino acids at
the end of p25 were made. pSV.p25.AD3 was digested with
BssHII, treated with Klenow, and digested with
BglII. Oligonucleotides with the sequences
5'-GATCGTAAGAAAGGTCGCAAAAAGGTCTAGA-3' and
5'-TCTAGACCTTTTTGCGACCTTTCTTAC-3' or
5'-GATCACCATCACCATCACCACGTCTAGA-3' and
5'-TCTAGACGTGGTGATGGTGATGGT-3' were annealed and ligated to
the prepared pSV.p25.AD3 vector to create pSV.p25.RKK and pSV.p25.H6,
respectively.
To determine whether the HFV Gag protein contains I domains, several
plasmids were constructed to express RSV-HFV chimeras. These constructs
were made by using PCR to amplify the desired sequences of HFV from the
plasmid pHSRV13. The oligonucleotides used in the PCR were designed to
incorporate BglII and BssHII restriction
endonuclease sites (underlined) flanking the HFV sequences. The
amplified product was digested with the respective enzymes and then
cloned into the plasmid pSV.G1P (7) in place of
the small fragment generated by double digestion with BglII
and BssHII. Amplifications of fragments for pSV.Bg.1,2,3,
pSV.Bg.1,2, and pSV.Bg.1 all used
5'-TTGAAGATCTGATGCTTTCTGGACAAAATTA-3' as the forward primer and
5'-TACTAGGCGCGCGTTAACACCCCTTGTTT-3',
5'-TACTAGGCGCGCTGATTTGGCCTAGGAGTTT-3', and
5'-TACTAGGCGCGCGGAAGATTATATCCTCCTTGA-3' as the
respective reverse primers. Amplifications of fragments for pSV.Bg.2
and pSV.Bg.2,3 both used
5'-TTGAAGATCTATCTAGTACTCAGAATCAAAAT-3' as the
forward primer and
5'-TACTAGGCGCGCTGATTTGGCCTAGGAGTTT-3' and 5'-GTCGCGCGCTTAGGATGATCGTTGGTTT CGGTT-3' as
the respective reverse primers. Finally, amplification of the
fragment for pSV.Bg.3 used 5'-TTGAAGATCTTCAAACTCCTAGGCCAAAT-3' as the
forward primer and 5'-GTCGCGCGCTTAGGATGATCGTTGGTTTCGGTT-3' as the
reverse primer.
Transfection and labeling of cells and immunoprecipitation of Gag
proteins.
COS-1 cells were transfected by the
DEAE-dextran/chloroquine method, as previously described
(36). Approximately 48 h after transfection, the cells
were labeled for 2.5 h with either
L-[4,5-3H(N)]leucine (150 µCi, 60 Ci/mmol)
or 50 µCi (>1,000 Ci/mmol) of
L-[35S]methionine (RSV-HFV chimeras). The
cells and growth medium from each labeled culture were separated and
mixed with lysis buffer containing protease inhibitors (36).
The Gag proteins were immunoprecipitated with polyclonal rabbit serum
against whole RSV (32) (reacts with MA, capsid [CA], NC,
and PR) or a combination of 
-RSV serum and polyclonal goat serum
against MLV CA. The immunoprecipitated proteins were resolved by
electrophoresis in sodium dodecyl sulfate (SDS)-12% polyacrylamide
gels and detected by fluorography (36).
Isopycnic sucrose gradient analysis.
To determine the
density of the particles produced by our various constructs,
transfected COS-1 cells were labeled for 8 h with either 500 µCi
of L-[4,5-3H(N)]leucine or 100 µCi of
L-[35S]methionine (RSV-HFV chimeras). The
growth medium was collected and centrifuged for 1 min at
15,000 × g to remove cellular debris before being
layered onto a 10 to 50% sucrose gradient. An internal control was
included in each gradient and consisted of either purified, unlabeled
Moloney MLV or labeled COS-cell-produced RSV particles of wild-type
density. The gradients were spun for 16 h at 83,500 × g in a Beckman SW41Ti rotor, and 0.6-ml fractions were
collected from the bottom of each tube. The relative amount of MLV in
each fraction was determined by the incorporation of [-32P]TTP during synthesis of DNA on a poly(A)
template as described previously (1). The amount of labeled
Gag protein in each fraction was determined by immunoprecipitating with
the appropriate antibody followed by SDS-polyacrylamide gel
electrophoresis (PAGE), fluorography, and densitometry of the
Gag-specific bands.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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When expressed in mammalian cells, the RSV Gag protein directs the production of virus-like particles. It is still able to produce particles when its M domain is replaced by that of the Src oncoprotein (37). However, the RSV Gag derivative Bg-Bs (Fig. 1A), which is truncated after residue 418 in CA, produces low amounts of particles (Fig. 1B) that are light in density (1.14 g/ml) relative to the wild type (Fig. 1C; see also reference 32). The low density of this mutant is due to the lack of I domains within the NC sequence, which are responsible for proper Gag-Gag interactions (33). Therefore, by fusing foreign sequences to Bg-Bs and assaying for restoration of normal density to the particles produced, I domains from other retroviral Gag proteins can be identified. This gain-of-function approach has been used to successfully locate two I domains in the NC region of the HIV Gag protein (1). To gain a better understanding of how I domains work, we initially set out to map the I domains of MLV and HFV Gag proteins and to characterize their important sequence features.
|
Identification of an MLV I domain.
Several chimeric proteins
were made to identify I domains in the MLV NC region (Fig. 1A). When
amino acid residues 314 to 538 from the MLV Gag protein were fused to
Bg-Bs to form BgM (Fig. 1A), efficient release of protein into the
medium was restored (Fig. 1B). Since this construct contains most of
the adjacent pol gene, including the sequences encoding the
MLV protease, Gag cleavage products were also evident, including the
chimeric capsid species p34CA (Fig. 1B, lanes 2). Protease
activity was not required for budding, however, since BgM.PR was
released just as efficiently (Fig. 1B, lanes 3).
When particles made from BgM were analyzed in sucrose gradients, they
were found to be of higher density (1.19 g/ml; Fig. 1C) than those from
Bg-Bs (Fig. 1C), indicating that amino acids either in the C terminus
of MLV Gag or in MLV Pol were able to provide I domain activity. The
particles from BgM were of slightly higher density than the control MLV
particles (1.16 to 1.18 g/ml), which is in agreement with previous
results for Env-less particles of RSV (37). To more
specifically map the region of MLV that contains this I domain
activity, we constructed truncations of BgM that ended after MLV amino
acid 401, 477, 500, or 520. Of these constructs, only the largest
(Bg.R521**) produced particles efficiently (Fig. 1B, lanes 4), and
these were of high density (1.19 g/ml; Fig. 1C). Low amounts of
particles (less than 10% of wild type) were obtained from Bg.D501**
and Bg.S402** (Fig. 1B, lanes 5 and 7) but were light in density (Fig.
1C). Bg.A478** did not produce enough particles to test in density
gradients. These data reveal the presence of at least one I domain in
the NC region of MLV Gag whereas similar analyses of RSV and HIV had revealed two (1, 8, 33).
Importance of basic residues for I domain function. The zinc fingers of Gag proteins have long been known to be dispensable for particle assembly and, in the case of MLV, particle density (13). To ensure that this was true for the RSV-MLV chimeras, we constructed zinc finger point mutations in the context of full-length BgM (Fig. 2A). In mutant Bg.C26/29S, the cysteine residues at positions 26 and 29 of MLV NC were replaced with serine residues, while in mutant Bg.Y28S, the tyrosine residue at position 28 was replaced with serine. Neither of these mutations reduced the number of particles released (Fig. 2B, lanes 4 and 5) nor the densities (1.18 g/ml) relative to wild type (Fig. 2C). This confirms that an intact zinc finger motif is not required for dense particle formation.
|
K8-R11, lacked two basic residues and one
acidic residue but had no effect on particle release (Fig. 2B, lanes 2)
or density (1.18 g/ml; Fig. 2C). The second deletion, Bg.R16-R23,
lacked eight amino acids, including four basic residues, and completely
(limit of detection, <2% of wild type) blocked particle release (Fig.
2B, lanes 3). The nature of the assembly defect of this construct is
not clear, but the lack of proteolytic processing in the cells (Fig.
2B, lanes 3) suggests that the mutant proteins were either not arriving
at the plasma membrane or not interacting with the mutant Gag-Pol
molecules once they arrived to allow protease activation. Because the
removal of the second set of basic residues had a detrimental effect on
particle assembly, we were unable to make a density determination for
this mutant and therefore cannot draw a conclusion from this result
about the contribution of these residues to the MLV I domain. However,
unpublished results (21a) indicate that MLV Gag proteins
with this deletion form particles of normal density and this, taken
together with the results from the C-terminal truncation mutants,
suggests that the MLV I domain maps somewhere between Gag residues 501 and 520 (residues 24 to 43 of NC).
To test the hypothesis that basic residues alone are sufficient for I
domain function, we investigated artificial sequences of basic residues
in our gain-of-function assay (Fig. 3).
Since CA is not required for budding and might actually interfere with the function of a small basic extension, we used the shorter RSV Gag
derivative named p25 (33). p25 contains only 180 Gag
residues and, like Bg-Bs, contains the M and L assembly domains but
lacks both I domains. Therefore, unless an I domain is added to its C
terminus, p25 releases few particles (down 10-fold from wild type; Fig.
3B, lanes 2), all of which exhibit lighter than normal density (1.14 g/ml; Fig. 3C). To decide which sequence of basic residues to use, we
examined the NC regions of RSV, HIV, and MLV. Within the regions
identified as I domains of these proteins, the following sequences are
found: PKKRK (RSV), PRKK (HIV), and PKKP (MLV). We wanted to use a
sequence similar but not identical to any of these, so we chose
the sequence RKKGRKK (p25.RKK). We also constructed a p25 chimera
with a stretch of six histidine residues, which is slightly basic but
does not resemble basic regions found within NC, at its C terminus
(p25.H6). Both chimeric proteins expressed well and released particles
(down 10-fold from wild type; Fig. 3B, lanes 3 and 4). As previously
described (33), the addition of residues 418 to 584 from RSV
Gag to the p25 truncation (p25.AD3) restored normal density to the
produced particles (1.18 g/ml; Fig. 3C). Similarly, p25.RKK (1.18 g/ml), but not p25.H6 (1.13 g/ml), was found to be capable of producing
dense particles (Fig. 3C). A portion of the extracellular Gag protein
from p25, p25.H6, and p25.RKK is found in the top fractions of the
gradient. We have previously shown (33) that the p25 Gag
proteins that sediment into the gradient (fractions 11 and 12, Fig. 3C)
are membrane enclosed and that the proteins at the top of a gradient containing particles from a NC construct (which also lacks I domains) are not particulate (data not shown). As expected, the upper material fails to shift in the gradients relative to the internal
control while the material that moves well into the gradient changes
density in a construct-dependent manner. It should be noted that the
particles formed by p25.RKK are more heterogeneous in density than
those formed by the BgM mutations (Fig. 1C and 2C). However, the
heterogeneity of the p25.RKK particles is only slightly more pronounced
than that of the p25.AD3 particles and the bulk of the peak is clearly
shifted relative to the peak of p25 particles. Thus, from this
experiment, it seems that a simple string of strongly basic
residues is sufficient to provide I domain function.
|
Absence of zinc fingers in HFV I domains. Although the experimental evidence shows that zinc finger motifs can be disrupted without inhibiting I domain function (13), there remained an apparent correlation between the number of I domains present in a given Gag protein and the number of zinc finger motifs. That is, our low-resolution mapping of I domains revealed two in RSV Gag and two in HIV Gag, but only one in MLV Gag. To eliminate the possibility that some aspect of the zinc finger other than zinc binding was contributing to I domain function, we decided to examine the HFV Gag protein, which lacks zinc fingers, for the presence of I domains.
Within the HFV NC sequence are three glycine-rich (GR) regions containing large numbers of basic residues (previously designated GR boxes I, II, and III; see reference 25). Since our previous experiments indicate that basic residues are likely to be important, we made Bg-Bs/HFV chimeric proteins that contained various combinations of these three regions (Fig. 4A). Although each RSV-HFV chimera expressed well, the amount of protein released into the media varied considerably (from 2 to 35% of wild type; Fig. 4B). In each case, however, enough extracellular material was available to perform sucrose density gradient analysis. When the entire NC region of HFV (amino acids 414 to 780) was fused after residue 418 of RSV Gag to form Bg.1,2,3, dense particles were produced (1.19 g/ml; Fig. 4C). This was also true when residues 414 to 581 (Bg.1,2, 1.19 g/ml) or only residues 414 to 540 (Bg.1) were added to Bg-Bs (Fig. 4C). These results show that an I domain is present within amino acids 414 to 540 of HFV Gag. To determine whether additional I domains were present in the HFV Gag sequence downstream of residue 540, we examined particles from Bg.2 (containing residues 523 to 581), Bg.3 (residues 574 to 780), and Bg.2,3 (residues 523 to 780) (Fig. 4C). Although particles from Bg.2 and Bg.3 were lighter than normal (1.16 g/ml), when the two regions were combined, the resultant protein (Bg.2,3) was able to form dense particles (1.19 to 1.22 g/ml). This result indicates the presence of an additional I domain downstream of the one contained in Bg.1. From these experiments, we can conclude that HFV NC contains at least two I domains in the absence of zinc finger motifs. Although the regions of HFV Gag that contain the interaction domains are also rich in basic residues, the importance of these residues for HFV I domain function requires further investigation.
|
Complementation analysis.
Previous experiments with the Gag
protein of RSV have shown that all types of mutants defective for
membrane binding can be rescued into particles by complementation, but
this requires the presence of functional I domains (2, 34,
37). In contrast, the unmyristylated form of the MLV Gag protein
cannot be rescued by functional MLV Gag, even when the single I domain
is intact (27). These observations suggest the possibility
that differences between the I domains of RSV and MLV are responsible
for the different rescue phenotypes. To test this hypothesis, we
examined the ability of a myristate-minus form of the BgM chimera to be
rescued. Because its I domain is derived from MLV, we predicted that
this chimeric protein would not be rescued into particles when its
membrane-binding domain (here derived from Src) was inactivated.
We used the protease-proficient form of BgM.M() and a
protease-deficient form of the rescuing molecule. Thus, the appearance
of Gag cleavage products in the medium would be an indicator of rescue.
) was expressed, it was unable to release particles on its
own (Fig. 5B, lanes 5) and, as expected
for a non-membrane-targeted Gag protein (36, 37), exhibited
reduced processing by the MLV protease (compare with Fig. 5B, lanes 4).
Upon coexpression of BgM.M() with a protease-deficient,
budding-competent MLV protein, M.M1.PR, both full-length products
could be observed in the cell lysates but only the MLV Gag precursor,
in the absence of any cleavage products, was evident in the media (Fig.
5B, lane 6). Similarly, a budding-competent RSV protein,
R.M1.PR, was unable to rescue BgM.M(), and again, only uncleaved
RSV Gag is seen in the medium (Fig. 5B, lane 7). Thus, it appears
that BgM.M() cannot interact with other Gag proteins to be rescued
into particles and therefore behaves like the unmyristylated MLV Gag
protein (27), consistent with the hypothesis that the
determinant of rescuability resides in the C terminus of Gag. The
trivial explanation that the myristate-minus form of BgM (and MLV Gag)
is globally misfolded and therefore not rescued for reasons unrelated
to MLV I domain function seems unlikely because mutants having the same lack of myristate in the context of the RSV-Src chimera can be rescued
with ease (37).
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(Fig. 5A), which lacks amino acids 122 to 336, including the entire L domain, but retains the M and I domains
(34). As shown in Fig. 5D (lanes 3), this molecule is
budding incompetent but is rescuable by full-length RSV Gag (Fig. 5D,
lanes 5). This well-characterized RSV L-domain mutation was introduced
into BgM to create chimera T10M.PR (lanes 4). When expressed by
itself, this recombinant, like T10C.PR, was unable to bud from the
cell (Fig. 5D, lanes 4). The presence of the strong membrane-binding
sequence of Src and the high proteolytic activity of the PR+ form (data
not shown) suggest that T10M is targeted to the plasma membrane. When
coexpressed with an assembly-competent molecule (M.M1.PR), T10M.PR
was readily rescued into particles (Fig. 5D, lanes 6), which further
indicates that it is not severely disrupted by the T10 deletion. This
evidence supports the idea that interactions among MLV Gag proteins
occur after the molecules are targeted to, and concentrated on, the
plasma membrane (see Discussion).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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I domains are essential for the production of retroviral particles of the proper density. The sequence characteristics and mechanism of action of I domains are, however, largely unknown. The data presented here show that a simple artificial string of basic residues is sufficient for I domain function. In addition, we provide evidence that I domains may play a role in the timing of initial Gag-Gag interactions and that RSV Gag proteins may interact earlier during assembly than MLV Gag proteins.
Mechanism of I domain function. I domains are currently thought to influence particle density by interacting with RNA and using it as a scaffold to facilitate the subsequent Gag-Gag protein interactions mediated by CA (1, 14, 16, 17). This model is supported by in vitro assembly data from RSV (6) and HIV Gag (6, 14) that shows formation of multimeric structures dependent on, and proportional in length to, added RNA. Although the genomic viral RNA is used during infection, it is not required for in vitro Gag multimerization (6, 14) or for the production of dense, Gag-only particles (36). Collectively, these results suggest that the nonspecific RNA binding activity of NC is involved in I-domain function. Further evidence that the Cys-His boxes, which are required for the specific incorporation of viral RNA into the particle (3), are not part of the I domain comes from studies of point mutations within the single zinc finger of MLV (13). The experiments reported here have extended this finding by showing that particle density is not affected by additional NC mutations in the context of RSV-MLV chimeras. They have also strengthened the above model by demonstrating that a simple string of basic residues, which should interact with RNA but should not exhibit binding specificity for the viral nucleic acid, can substitute for NC's density-determining properties. However, while it is clear that basic residues can replace the RSV I domains, it may be that not just any basic array will suffice.
Conservation of RNA-mediated assembly. In addition to identifying the functional component of I domains, we also sought to determine the extent to which I domains are maintained between distantly related retroviruses. Although spumavirus NC sequences do not contain zinc fingers, they do contain three GR boxes which have been implicated in nucleic acid binding (38). Since sequence inspection is not informative for identifying I domains, we used a gain-of-function approach to identify the presence of at least two I domain equivalents within HFV Gag. The location of the HFV I domains is consistent with previous reports indicating that GR box 1 is the primary determinant of HFV NC nucleic acid binding (38). RNA binding by GR box 2 or 3 alone is drastically reduced (38) but, when present together, they cooperate sufficiently to provide I domain function.
Spumaviruses have been observed to exhibit strong nuclear localization in infected cells (10, 15). Although our RSV-HFV chimeras were capable of producing dense particles, they did not exhibit the strong nuclear localization (data not shown) previously seen for HFV NC sequences (26). We considered the possibility that the myristylated amino terminus of our chimeras was a dominant plasma membrane-targeting signal and made myristate-minus forms of our chimeric spumavirus constructs. Again, however, strong nuclear localization was not observed by either immunofluorescence or cell fractionation (data not shown). The difference in nuclear localization may be the result of using different expression systems (vaccinia-virus-expressed [26] versus COS-cell-expressed chimeras) or may be related to the fact that, during infection, nuclear localization is transient (26). However, the relevance of nuclear localization is questionable since recent results indicate that nuclear targeting is not essential for HFV infectivity (38). Regardless of the issue of nuclear localization, our finding of sequences in the NC region of HFV that can provide the interactions necessary to restore dense particle production to an RSV mutant Gag protein suggests that spumaviruses, even in the absence of zinc finger domains, may utilize RNA during assembly to initiate protein-protein contacts.Timing of initial Gag-Gag interactions. Although I domains play a major role in the formation of proper Gag-Gag interactions, the location and timing of the initial contacts remain unknown. Because type B and D retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble electron-dense cores in the cytoplasm of an infected cell (9), we know that Gag proteins from these viruses are able to interact prior to membrane transport. However, since a single point mutation in the matrix region of M-PMV Gag causes core assembly to take place at the plasma membrane rather than in the cytoplasm (23), it appears that the membrane-binding and targeting domain, rather than the I domain, determines the cellular site of assembly. Although the Gag proteins of C-type retroviruses, such as RSV, do not become visible until after their localization to the plasma membrane (4, 5), it is quite possible that they begin to interact prior to membrane transport but in a manner not visible by electron microscopy. In fact, complementation analyses showing rescue of membrane-binding mutants suggest that RSV Gag proteins may interact prior to membrane localization (34, 37). In contrast to RSV, unmyristylated MLV Gag proteins, which also exhibit C-type plasma membrane assembly, are unable to be rescued in vivo (27). This indicates either that MLV Gag proteins do not interact with each other in the cytoplasm or that any pre-membrane-localization interactions are not of sufficient strength for rescue to occur.
Although the observations that cytoplasmic MLV and RSV Gag molecules have different rescue phenotypes were made some time ago, the molecular basis of those phenotypes remains unexplained. Our results map this difference to the C terminus of Gag. Membrane-binding mutants having the RSV C terminus can be rescued (37) while those with MLV C-terminal sequences cannot. Since the inability to be rescued is a negative result, a number of trivial explanations could account for our observations. As has been previously suggested for unmyristylated MLV (27), the lack of rescue of our RSV-MLV chimera could be due to low expression levels, rapid protein degradation and/or misfolding, or an inability of the protein to interact with the rescuing Gag molecule. Low expression or rapid degradation of BgM.M()
does not appear to be a problem since large amounts (i.e., similar to
M.M1 levels) of this protein are present in the cell lysates. Although
BgM.M() could be globally misfolded, this is unlikely because loss of
myristate is not known to severely disrupt the structure of other Gag
proteins. For example, myristate-minus Gag proteins of M-PMV are still
able to associate into cytoplasmic cores (24) and studies
with myristylation inhibitors have shown that HIV particles can form
even when only a few of the Gag proteins are myristylated
(19). Likewise, even RSV Gag proteins that have become
dependent on the Src membrane-binding sequence (e.g., R.M1) can be
rescued when myristate is absent (37). Furthermore, chimeras
containing the large T10C deletion (i.e., T10M) are readily rescued,
indicating that the BgM chimera is not overly sensitive to mutations.
This plasticity of Gag is emphasized further by the fact that BgM can
be packaged with either MLV or RSV Gag, even though BgM contains a
highly distorted capsid sequence, and this represents the first
evidence for copackaging of Gag proteins from different retroviruses.
Experiments demonstrating that copackaging of MLV and RSV Gag proteins
requires that each protein contain the same membrane-binding domain
will be presented elsewhere (2). Thus, the simplest
interpretation of our results is that the MLV Gag protein (but not that
of RSV) requires concentration at the membrane in order for Gag-Gag
interactions to occur.
One of the most striking differences between the C termini of RSV and
MLV is the presence of two readily identified I domains in the former
and one in the latter. This observation leads us to hypothesize that
there may be a dosage effect in which two I domains provide
interactions strong enough to allow Gag proteins to associate in the
cytoplasm. The presence of a single I domain would allow interactions
only after concentration at the plasma membrane. Consistent with this
idea is data showing that the myristate-minus form of the Gag protein
of spleen necrosis virus (SNV) cannot be rescued into particles by
wild-type SNV Gag (31). SNV is closely related to MLV
(~40% identity) and also has a single Cys-His box. The ultimate test
of our hypothesis, however, will be to find mutants of MLV, perhaps
containing a duplicated NC region with two I domains, that can be
rescued when myristate is removed.
| |
ACKNOWLEDGMENTS |
|---|
This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL (A.R.), by NIH grants to J.B.B. (training grant CA60395) and J.W.W. (CA47482), and by a grant from the American Cancer Society to J.W.W. (FRA-427).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Dr., P.O. Box 850, Hershey, PA 17033. Phone: (717) 531-3528. Fax: (717) 531-6522. E-mail: jwills{at}psu.edu.
Present address: Invitrogen Corporation, Carlsbad, CA 92121.
Present address: Department of Molecular Biology, The Scripps
Research Institute, La Jolla, CA 92037.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Virology, November 1998, p. 9034-9044, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Sandefur, S., Smith, R. M., Varthakavi, V., Spearman, P.
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Cimarelli, A., Luban, J.
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Buchschacher, G. L. Jr., Yu, L., Murai, F., Friedmann, T., Miyanohara, A.
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Lee, E.-g., Yeo, A., Kraemer, B., Wickens, M., Linial, M. L.
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Cen, S., Huang, Y., Khorchid, A., Darlix, J.-L., Wainberg, M. A., Kleiman, L.
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Bennett, R. P., Wills, J. W.
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